A piRNA-lncRNA regulatory network initiates responder and trailer piRNA formation during mosquito embryonic development

Betting, Valerie; Joosten, Joep; Halbach, Rebecca; Thaler, Melissa; Miesen, Pascal; Rij, Ronald P. van

doi:10.1101/2020.03.23.003038

Cited by 7 publications

(11 citation statements)

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“…As viral RNA levels are virtually unchanged between all viruses, reduced responder piRNA production cannot result from differences in the amount of available substrate (Figure S4H). Weak responder piRNA production was observed in two seed mutants (Mut 1-3 and Mut 4-6) and a slice site mutant (Mut10-11), suggesting that low level slicing may occur even in the absence of full complementarity in the seed region or the slice site, in line with earlier findings in Drosophila (8), mice (47) and mosquitoes (48). Altogether, these data show that slicing by the ping-pong partners Ago3 and Piwi5 is required for the production of responder piRNAs from the viral reporter.…”

Section: Resultssupporting

confidence: 86%

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA inAedes aegyptimosquitoes

Joosten

Overheul

Rij

et al. 2020

Preprint

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ABSTRACTPIWI interacting (pi)RNAs are small RNAs mostly known to protect the genomes of animal germlines against transposable elements. In Drosophila, piRNA-mediated cleavage of transposon RNA triggers the release of a responder piRNAs via the ping-pong amplification cycle. Responder piRNA 3’ end formation by the endonuclease Zucchini is coupled to the production of downstream trailer piRNAs, expanding the repertoire of piRNAs that target transposons. Intriguingly, in Aedes aegypti mosquitoes, somatic piRNAs are produced from viral RNA, implying a role of viral (v)piRNAs in antiviral immunity. Knowledge on how vpiRNA 3’ ends are formed and whether viral RNA is subjected to trailer piRNA production, however, is lacking. To address these questions, we analyzed small RNA sequencing libraries from Ae. aegypti cells. We found that virus- and transposon-derived piRNAs have sharply defined 3’ ends, and that uridine residues are enriched directly downstream of dominant piRNA sequences, both of which are characteristic features of Zucchini-like cleavage of precursor piRNAs. Next, we designed a piRNA reporter system based on Sindbis virus recombinants that harbor target sites for abundant endogenous piRNAs. These piRNAs guide cleavage of the viral RNA, which is subsequently processed into abundant responder piRNAs. Using this reporter virus system, we identified the Ae. aegypti orthologs of Zucchini, which is required for sharp 3’ end formation of responder piRNAs, and Nibbler, a 3’-5’ exonuclease involved in the trimming of a subset of piRNAs and miRNAs. Furthermore, we found that cleavage of viral RNA triggers the production of trailer piRNAs, thus expanding the piRNA sequence pool that is able to target viral RNA. Our results have important implications for understanding how autonomous piRNA production from viral RNA can be triggered by just a few cleavage events by genome-encoded piRNAs.

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Section: Resultssupporting

confidence: 86%

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA inAedes aegyptimosquitoes

Joosten

Overheul

Rij

et al. 2020

Preprint

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“…The identified interactors were depleted by double-stranded RNA (dsRNA)-mediated KD, followed by small RNA Northern blotting to detect piRNAs produced from endogenous loci (histone H4 mRNA and propiR1) and exogenous viral RNA (SINV). The endogenous piRNA propiR1 exists as two isoforms of 27 and 30 nt that target a specific long non-coding RNA during early embryonic development (Betting et al, 2020). In line with our previous work, KD of the ping-pong partners Ago3 and Piwi5 resulted in a strong depletion of piRNAs derived from SINV RNA and histone H4 mRNA (Figures 2A, S3D, and S3E) (Miesen et al, 2015;Girardi et al, 2017), whereas Piwi5 KD caused a reduction of the short 27-nt propiR1 isoform that is associated with Piwi5 (Betting et al, 2020).…”

Section: Resultsmentioning

confidence: 99%

“…aegypti, the pathway has been implicated in diverse processes ranging from antiviral defense See also Figure S6. (Miesen et al, 2015;Schnettler et al, 2013;Vodovar et al, 2012;Morazzani et al, 2012) and immune memory (Tassetto et al, 2019;Suzuki et al, 2020;Crava et al, 2021) to gene regulation (Girardi et al, 2017) and embryonic development (Halbach et al, 2020;Betting et al, 2020). However, knowledge of piRNA biogenesis and effector mechanisms in Ae.…”

Section: Discussionmentioning

confidence: 99%

“…For example, the vector mosquito Aedes aegypti expresses an expanded PIWI protein repertoire of seven members, several of which are expressed in somatic tissues, suggesting that the pathway has additional functions besides transposon silencing (Miesen et al, 2016;Campbell et al, 2008;Lewis et al, 2016). Indeed, the Aedes piRNA pathway has been implicated in noncanonical processes, such as antiviral defense (Morazzani et al, 2012;Miesen et al, 2015;Vodovar et al, 2012;Schnettler et al, 2013), endogenous viral element (EVE)-mediated immune memory (Tassetto et al, 2019;Suzuki et al, 2020;Crava et al, 2021), mRNA elimination during embryonic development (Halbach et al, 2020;Betting et al, 2020), and gene regulation (Girardi et al, 2017;Arensburger et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes

et al. 2021

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As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.

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“…aegypti [ 96 , 97 , 98 , 99 ], more of the Ae. aegypti piRNAs have been mapped to protein-coding genes abundantly expressed in the soma [ 100 , 101 ].…”

Section: Genome Transposon Composition and Germline Specificity Ofmentioning

confidence: 99%

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways

Gamez

Srivastav

Akbari

et al. 2020

Cells

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Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.

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A piRNA-lncRNA regulatory network initiates responder and trailer piRNA formation during mosquito embryonic development

Cited by 7 publications

References 70 publications

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA inAedes aegyptimosquitoes

Endogenous piRNA-guided slicing triggers responder and trailer piRNA production from viral RNA inAedes aegyptimosquitoes

PIWI proteomics identifies Atari and Pasilla as piRNA biogenesis factors in Aedes mosquitoes

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways

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